Understanding the molecular mechanisms that regulate developmental potential of a cell is a fundamental question that has great implications in regenerative medicine. Cells within 1-, 2-, and 4-cell stage embryos feature the highest developmental potential, termed totipotency. By contrast, embryonic stem cells are pluripotent with cell lineage restriction. However, while the genetic and epigenetic circuits governing pluripotency are well understood, those governing totipotency are poorly understood. This is partly due to the limited cell numbers available from early preimplantation embryos, hampering genetic screening of totipotency regulators. Recently, it has been shown that a small population of cells within mouse embryonic stem cells (mES cells) has totipotent characteristics, termed 2C-like cells (Macfarlan et al, 2012). Taking advantage of this discovery, I propose to perform an unbiased genome scale genetic screen for totipotency regulators using a CRISPR-Cas9 based approach. I plan to transduce a pooled CRISPR- Cas9 knockout library to a fluorescent mES reporter cell line of 2C-like cells. 2C-like cell populations will be sorted by Fluorescence-Activated-Cell Sorter (FACS), and then high-throughput sequencing will be used to identify integrated sgRNAs. In parallel, DNA from unsorted mES cells will also be sequenced. By comparing the sequencing results of these two groups, genes that activate or repress the 2C-like state can be identified. Next, I will focus on the epigenetic factors and transcriptional regulators identified from the screen to understand the gene regulatory circuit governing totipotency. For each candidate, ChIP-seq will be performed in 2C-like cells and non-2C-like ES cells. The results will be compared with RNA-seq analysis obtained from control and knockout cells, which would reveal direct targets of each totipotency regulator. To expand the findings from mES cells in vitro to in vivo, I will either knock-down or over- express the candidate genes in pre-implantation embryos by injecting siRNA or mRNA into the zygote (1- cell stage embryo). The impact on early development will be investigated. In parallel, RNA-seq will be performed using 2- or 8-cell stage embryos to examine establishment or maintenance of the totipotent transcriptome. Completion of the proposed study will greatly advance our understanding of totipotency and developmental biology.